Isolation and detailed characterization of the components constituting the cytochrome b5 and P-450 systems of mammalian liver endoplasmic reticulum are the broad objectives of this proposal. Among the specific objectives of the proposed study are the following: 1) elucidation of the entire amino acid sequence of the predominant form of cytochrome b5 with a detailed study of the amino acid sequence of cytochrome b5 in normal and pathological human liver; 2) extension and refinement of the purification techniques used in the isolation of cytochrome P-450 from human, rat, and pig liver microsomes. This will include studies of the spectral properties, molecular weight, and amino acid composition; 3) collaborative investigation of the properties and primary structure of the human cytochrome b5 reductase; 4) joint studies, on the reconstitution of the polyfunctional oxidase system with emphasis towards drug metabolism, and the conversion of aromatic hydrocarbons into mutagens; 5) continuation of collaborative studies on the isolation of monospecific antisera to cytochrome b5 and P-450 in various animals, for use in the immunological "sandwich" technique to localize these proteins in normal and pathological tissues. At present, using this method we can demonstrate cytochrome b5 in paraffin sections of rat liver. Study of these proteins is important because they constitute the microsomal oxidase system which hydroxylates endogenous and exogenous lipophilic compounds, drugs and hydrocarbons. The broad substrates specificity, as well as its unpredictable inducibility, implicate this system in adverse drug reactions and pharmacological synergism. At present, these phenomena are poorly understood. Essential to their understanding are the isolation and delineation of the molecular features of the hemoproteins and reductases constituting this system. Moreover, reconstitution of the hydroxylation system of lipophilic compounds in vitro should also provide an ideal model system to determine the specific pathways by which biotransformation of drugs and potential carcinogens occur in animals and in man. BIBLIOGRAPHIC REFERENCES: Cinti, D.L. and J. Ozols, (1975) Binding of cytochrome b5 to rat liver microsomes: Effect on N-denethylation reactions. Biophys. Biochim. Acta, 410:32-44. Ozols, J. (1976) Microsomal electron transport in human hepatic membran (Text Truncated - Exceeds Capacity)